Cell culture is a technology of taking tissues of skin, lung or kidney or cells such as lymphocytes and leukemia cells out of a living body and growing and keeping them alive in vitro.
With the development of cell culture technology in recent years, its application not only for the elucidation of life phenomena but also for the manufacture of virus vaccine, the production of biological medicines, the gene therapy or regenerative medical therapy and the immuno-cell therapy has been studied and implemented.
The conventional procedure of cell culture performed in the immuno-cell therapy is explained with reference to FIG. 14.
In the conventional cell culture method, cells were cultured while transferring (subculture) the cells to another cell culture container having a larger volume with the proliferation of the cell.
Specifically, as shown in the figure, at first, 30 cc of a culture medium (here, it contains 107 immune cells) is put in a cell culture flask (hereinafter simply referred to as “flask”).
In this culture, the temperature, carbon dioxide concentration (CO2) and humidity are set at 37° C., 5% and 95%, respectively.
Three days after, the volume of the culture medium in the flask was increased in an amount of 30 cc to 60 cc, and four days after, the volume of the culture medium was further increased in an amount of 60 cc to 120 cc. In view of the relationship between the flask volume and the cell density, the culture medium is transferred to another cell culture container. Here, two flasks are newly provided, and subculture is conducted such that each flask is charged with 40 cc of the culture medium.
When the culture medium in each flask is increased from 40 cc in an amount of 40 cc to become 80 cc, then, the culture medium in each of the three flasks is transferred to one cell culture bag (hereinafter simply as a “bag”). 240 cc (80 cc×3) of the culture medium, cells being cultured and 1000 cc of a fresh culture medium are enclosed in this bag, and cultured for two days. Furthermore, in respect of the relationship between the volume of the bag and the cell density, a bag is newly provided and subculture is conducted such that each bag is charged with 620 cc of the culture medium.
Then, in each bag, 500 cc of a fresh culture medium is added to the 620 cc of the culture medium to make the total volume thereof 1120 cc, and culture is conducted. Thereafter, two bags are newly provided, and subculture is conducted such that each bag is charged with 560 cc of the culture medium.
As mentioned above, by sequentially transferring a culture medium to a culture container having a larger volume or by increasing the number of a container, an appropriate culture environment can be maintained.
The reason for transferring cells is as follows. When the cell density is low when culture is started, proliferation of cells is suppressed. Therefore, when cells are cultured on a certain scale, it is common to culture the cell while repeating subculture so that the cell density can be maintained appropriately.
However, this method makes the procedure complicated since part cells being cultured has to be transferred to a new flask or bag (or a dish) whenever subculture is conducted. In addition, this method has a higher degree of probability of being contaminated with various bacteria.
Under such circumstances, a method can be conceived in which cells are caused to proliferate in a bag, and when the cells are proliferated, a new bag filled with a fresh culture medium is connected to attain uniformity. By this method, the risk of being contaminated by various bacteria can be lowered.
However, it appears that this procedure takes a certain period of time until uniformity is attained. The larger the internal volume of the culture bag, the longer the time required for attaining uniformity. The procedure becomes complicated, although it is less complicated as compared with the procedure in the open system. In addition, the quantity of cell culture equipment to be consumed is increased.
Under such circumstances, various technologies are proposed to maintain an appropriate culture environment without conducting subculture. For example, a cell culture bag has been disclosed which is provided with prohibiting members by which the bag is partitioned so that the circulation of the culture medium in the bag is prohibited (see Patent Document 1, for example).
In this technology, the region in the bag where culture is actually conducted can be gradually extended. Therefore, culture can be conducted continuously within the same bag from the start to the end. In this technology, the fear of contamination with various bacteria can be eliminated, culture can be conducted taking into consideration the cell density at the time of starting culture or during culture depending on the proliferation capability of cells, and cells can be cultured efficiently by means of simple equipment.
Also disclosed is a cell culture system comprising an external partition member (a partitioning clamp) for dividing a single bag-like enclosure into sub-compartments (see Patent Document 2, for example).
According to this culture system, a plurality of sub-compartments can be provided by squeezing part of the bag-like enclosure by means of the external partition member. Therefore, it is possible to provide means to proliferate cells to a number enough to be introduced into the production system under conditions which provide a small-scale starting environment for ensuring the viability of cells and a necessary environment which is enlarging in scale.
Also disclosed is a culture container provided with an elastically deformable tubular culture container and a clip capable of squeezing the culture container at an arbitral position (see Patent Document 3, for example).
According to this culture container, it is possible to increase the internal volume of the culture container by shifting the position of the clip according to the growth of cells.
Patent Document 1: JP-A-2000-125848
Patent Document 2: Japanese Patent No. 2981684 (page 5)
Patent Document 3: JP-A-2004-89136 (page 5, paragraph [0021] and FIG. 3)